Methods of protein clips recovery

ABSTRACT

The disclosed methods are directed to detecting polypeptide fragments (“clips”) of parental polypeptides. Parental polypeptides and clips are optionally denatured and then fractionated using a matrix. After a first elution (high molecular weight fraction), an additional elution step retrieves a low molecular weight fraction containing clips. If the clips are an appropriate size for the targeted detection method, such as mass spectrometry, then analysis of this fraction proceeds separately from the high molecular weight fraction, or the clips fraction is mixed with the proteolyzed high molecular weight fraction before analysis. However, if the clips are too large for the intended analytical method(s), then the clips are also proteolyzed. The digested high molecular weight and low molecular weight fractions can be analyzed separately or combined. Analysis of combined samples favors clip quantitation because the clips are analyzed together with the remaining counterpart of the parental polypeptide.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/527,402 filed Jun. 30, 2017, which is incorporated inits entirety by reference herein.

SEQUENCE LISTING

The present application is being filed with a sequence listing inelectronic format. The sequence listing provided as a file titled,“A-2141-US-NP_sequence_listing_ST25.txt”, created Jun. 8, 2018, and is262 KB in size. The information in the electronic format of the sequencelisting is incorporated herein by reference in its entirety.

FIELD

The presented subject matter relaters to the field of protein analysis.Specifically, the presented subject matter relates to the preparation ofsamples for the detection of polypeptides comprised therein, such astherapeutic polypeptides, that are clipped (fragmented) duringmanufacturing or storage.

BACKGROUND

Fragmentation (clipping) of polypeptides (creating polypeptide “clips”),such as clips of therapeutic polypeptides, can occur duringmanufacturing process or storage due to chemical and enzymatic causes.Identifying and quantifying clips can be challenging, because clips areoften lost during sample preparation for peptide mapping typeexperiments. Clipping is undesirable because it represents degradationof the parental polypeptide, and in the case of therapeuticpolypeptides, a possible reduction in potency.

There is a substantial need in the art to provide reliable methods forsample preparation and detection of clips found therein.

SUMMARY

Described herein are methods that can recover clips of polypeptides,such as therapeutic polypeptides, by separating the clips and theirintact polypeptide counterparts; these clips are often lost duringpreparation of compositions comprising the polypeptide for analysis,such as peptide mapping analysis. Such preparation methods facilitateddetection of these clips.

In a first aspect, disclosed herein are methods of detecting at leastone fragment of a polypeptide in a sample (e.g., a clip of thepolypeptide), comprising:

-   -   (a) fractionating the sample, comprising:        -   a. loading the sample onto a size exclusion chromatographic            matrix;        -   b. applying a volume of a first elution buffer to the            matrix, wherein the polypeptide is eluted into a first            fraction; and        -   c. applying a volume of a second elution buffer to the            matrix, wherein the at least one fragment is eluted into a            second fraction;    -   (b) treating the first fraction with a proteolytic enzyme; and    -   (c) optionally, combining the treated first fraction with the        second fraction, and    -   (d) detecting the polypeptide in the first fraction and        detecting the at least one fragment in the second fraction.

In a second aspect, disclosed herein are methods detecting at least onefragment of a polypeptide (e.g., a clip of the polypeptide) in a sample,comprising:

-   -   (a) fractionating the sample, comprising:        -   i. loading the sample onto a centrifugal device comprising a            molecular weight cutoff filter; and        -   ii. processing the sample such that the sample passes            through the cutoff filter such that the polypeptide is            retained on the filter to create a first fraction and the at            least one fragment that passes through the filter is            retained as a second fraction;    -   (b) treating the first fraction with a proteolytic enzyme;    -   (c) optionally, combining the treated first fraction with the        second fraction; and    -   (d) detecting the polypeptide in the first fraction and the        fragments in the second fraction.

In these first two aspects, the methods can further comprise, after step(a), treating the second fraction with a proteolytic enzyme. Thepolypeptide can be a therapeutic polypeptide, such as, for example, oneselected from the group consisting of an antibody or antigen-bindingfragment thereof, a derivative of an antibody or antibody fragment, anda fusion polypeptide. For example, the therapeutic polypeptide can be anantibody, wherein the antibody is selected from the group consisting ofinfliximab, bevacizumab, cetuximab, ranibizumab, palivizumab,abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab,alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab,altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab,arcitumomab, aselizumab, altinumab, atlizumab, atorolimiumab,tocilizumab, bapineuzumab, basiliximab, bavituximab, bectumomab,belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab,bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine,blinatumomab, blosozumab, brentuximab vedotin, briakinumab, brodalumab,canakinumab, cantuzumab mertansine, cantuzumab mertansine, caplacizumab,capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab,certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab,clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab,crenezumab, cr6261, dacetuzumab, daclizumab, dalotuzumab, daratumumab,demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab,duligotumab, dupilumab, ecromeximab, eculizumab, edobacomab,edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab,enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab,epitumomab cituxetan, epratuzumab, erenumab, erlizumab, ertumaxomab,etaracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab,faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab,ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab,foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab,gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab,girentuximab, glembatumumab vedotin, golimumab, gomiliximab, gs6624,ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab,imgatuzumab, inclacumab, indatuximab ravtansine, infliximab,intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab,itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab,lemalesomab, lerdelimumab, lexatumumab, libivirumab, ligelizumab,lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab,lumiliximab, mapatumumab, maslimomab, mavrilimumab, matuzumab,mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab,mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox,muromonab-cd3, nacolomab tafenatox, namilumab, naptumomab estafenatox,narnatumab, natalizumab, nebacumab, necitumumab, nerelimomab,nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab,ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab,onartuzumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab,oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab,panitumumab, panobacumab, parsatuzumab, pascolizumab, pateclizumab,patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab,pidilizumab, pintumomab, placulumab, ponezumab, priliximab, pritumumab,PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ramucirumab,ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab,rituximab, robatumumab, roledumab, romosozumab, rontalizumab,rovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetide,secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab,simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab,sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumabtetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox,tefibazumab, telimomab aritox, tenatumomab, tefibazumab, teneliximab,teplizumab, teprotumumab, tezepelumab, TGN1412, tremelimumab,ticilimumab, tildrakizumab, tigatuzumab, TNX-650, tocilizumab,toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07,tregalizumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, urelumab,urtoxazumab, ustekinumab, vapaliximab, vatelizumab, vedolizumab,veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab,vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab,ziralimumab, zolimomab aritox, and those antibodies shown in Table 1. Inother embodiments, the therapeutic polypeptide is selected from thegroup consisting a glycoprotein, CD polypeptide, a HER receptorpolypeptide, a cell adhesion polypeptide, a growth factor polypeptide,an insulin polypeptide, an insulin-related polypeptide, a coagulationpolypeptide, a coagulation-related polypeptide, albumin, IgE, a bloodgroup antigen, a colony stimulating factor, a receptor, a neurotrophicfactor, an interferon, an interleukin, a viral antigen, a lipoprotein,calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumornecrosis factor-alpha and -beta, enkephalinase, mousegonadotropin-associated peptide, DNAse, inhibin, activing, an integrin,protein A, protein D, a rheumatoid factor, an immunotoxin, a bonemorphogenetic protein, a superoxide dismutase, a surface membranepolypeptide, a decay accelerating factor, an AIDS envelope, a transportpolypeptide, a homing receptor, an addressin, a regulatory polypeptide,an immunoadhesin, a myostatin, a TALL polypeptide, an amyloidpolypeptide, a thymic stromal lymphopoietin, a RANK ligand, a c-kitpolypeptide, a TNF receptor, and an angiopoietin, and biologicallyactive fragments, analogs or variants thereof. In these aspects, thefragments (e.g., clips) are 0.3 kD to about 50 kD, or, for example about3 amino acids to about 455 amino acids. Furthermore, the proteolyticenzyme in any aspect or sub-aspect can be trypsin. The matrix cancomprise a gel filtration matrix. The gel filtration matrix can be, forexample, cross-linked dextran. In any case, the matrix can be preparedin a column before performing step (a). If the matrix is prepared in acolumn, the column can be suitable for gravity flow and/or centrifugalassisted flow. In yet other sub-aspects, the first elution buffer andthe second elution buffer can be the same; for example, they cancomprise 100 mM Tris and 50 mM methionine, pH 7.5. The volume of thefirst elution buffer can be greater than the second elution buffer. Insome sub-aspects, the methods can further comprise, before performingstep (a), performing at least one step selected from the groupconsisting of polypeptide alkylation, polypeptide reduction, andpolypeptide denaturation, or any combination thereof. The methods canalso further comprising detecting the polypeptide and the fragments(e.g., the clips). Such detecting can comprise, as an example, massspectrometry.

In a third aspect, disclosed herein are sample prepared by any of thefirst two aspects and their related sub-aspects.

In a fourth aspect, disclosed herein are methods of detecting at leastone fragment of a glycoprotein in a sample, comprising:

-   -   (a) denaturing, reducing, and alkylating the glycoprotein and        fragments in the sample;    -   (b) fractionating the sample, comprising,        -   a. loading the sample onto a cross-linked dextran gel            filtration column, wherein the cross-linked dextran gel that            has a fractionation range of about 1000-5000 Da;        -   b. applying a volume of a first elution buffer comprising            100 mM Tris and 50 mM methionine, pH 7.5 to the column,            wherein the glycoprotein is eluted into a first fraction;            and        -   c. applying a volume of second elution buffer comprising 100            mM Tris and 50 mM methionine, pH 7.5 to the column, where            the at least one fragment is eluted into the second            fraction;    -   (c) treating the first fraction with trypsin;    -   (d) combining the treated first fraction with the second        fraction; and    -   (e) detecting the glycoprotein in the first fraction and the at        least one fragment in the second fraction.

In a sub-aspect of this fourth aspect, the methods can further comprise,after step (b), treating the second fraction with trypsin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic depicting a method to collect polypeptide clipsfor detection.

FIG. 2 shows peptide mapping detected by mass spectrometry. FIG. 2Ashows the elution of the parental polypeptide, while FIG. 2B shows theelution of clipped polypeptides of the parental polypeptide.

DETAILED DESCRIPTION

Described herein are methods that can recover clips of polypeptides(such as therapeutic polypeptides) which are often lost during standardprotocols directed to sample preparation. The disclosed methods separatethe clips and their intact “parental” polypeptide counterparts. Afterbeing collected separately, the collected parental polypeptides aresubjected to enzymatic digestion, and the collected clips can be (1)mixed (if desired) with the digest of parental polypeptide if the clipsare small enough for analysis (depending on the analytical technique);or (2) subjected to enzymatic digestion before being mixed (if desired)with the digest of the parental polypeptide if the clips are too largeto be analyzed directly. The disclosed methods solve the problem ofclips being lost during sample preparation, such as those preparativemethods used for peptide mapping-type experiments.

The method is illustrated in FIG. 1. Referring to FIG. 1, a parentalpolypeptide and fragments thereof (“clips”) are separated by molecularweight cutoff filters, gel filtration or size exclusion-type columns 1,2. High molecular weight species elute earlier and are collected first3. The high molecular weight fraction is then digested with one or moreproteolytic enzymes 4. The low molecular weight species fraction 2, 5,which usually contains any clips, elutes later and can be collectedseparately 6, 7. The low molecular weight fraction is evaluated todetermine if the clips are an appropriate size for analysis 8. If theclips in this fraction are an appropriate size, then the fraction can beanalyzed, separately from the high molecular weight fraction 2, or mixedwith the digested high molecular weight fraction 9 and then analyzed.However, if the clips are too large for the intended analyticalmethod(s), then this fraction is also digested with one or moreproteolytic enzymes 10. Again, the digested high molecular weightfraction and the digested low molecular weight fraction can be analyzedseparately, or combined 11. Analysis of combined samples is simple andfavors clip quantitation because the clips are analyzed together withthe remaining counterpart of the parental polypeptide.

Definitions

Both the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictive.The use of the singular includes the plural unless specifically statedotherwise. The use of “or” means “and/or” unless stated otherwise. Theuse of the term “including”, as well as other forms, such as “includes”and “included”, is not limiting. Terms such as “element” or “component”encompass both elements and components comprising one unit and elementsand components that comprise more than one subunit unless specificallystated otherwise. The use of the term “portion” can include part of amoiety or the entire moiety. When a numerical range is mentioned, e.g.,1-5, all intervening values are explicitly included, such as 1, 2, 3, 4,and 5, as well as fractions thereof, such as 1.5, 2.2, 3.4, and 4.1.

“About” or “˜” means, when modifying a quantity (e.g., “about” 3 mM),that variation around the modified quantity can occur. These variationscan occur by a variety of means, such as typical measuring and handlingprocedures, inadvertent errors, ingredient purity, and the like.

“Comprising” and “comprises” are intended to mean that the formulationsand methods include the listed elements but do not exclude otherunlisted elements. The terms “consisting essentially of” and “consistsessentially of,” when used in the disclosed methods include the listedelements, exclude unlisted elements that alter the basic nature of theformulation and/or method, but do not exclude other unlisted elements.So a formulation consisting essentially of elements would not excludetrace amounts of other elements, such as contaminants from any isolationand purification methods or pharmaceutically acceptable carriers (e.g.,phosphate buffered saline), preservatives, and the like, but wouldexclude, for example, additional unspecified amino acids. The terms“consisting of” and “consists of” when used to define formulations andmethods exclude more than trace elements of other ingredients andsubstantial method steps for administering the compositions describedherein. Embodiments defined by each of these transition terms are withinthe scope of this disclosure.

“Antibodies” (Abs) and the synonym “immunoglobulins” (Igs) areglycopolypeptides having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific antigen,immunoglobulins include both antibodies and other antibody-likemolecules that lack antigen specificity. Polypeptides of the latter kindare, for example, produced at low levels by the lymph system and atincreased levels by myelomas. Thus, the term “antibody” or “antibodypeptide(s)” refers to an intact antibody, an antibody derivative, anantibody analog, a genetically altered antibody, an antibody having adetectable label, an antibody that competes for specific binding with aspecified antibody, or an antigen-binding fragment (e.g., Fab, Fab′,F(ab′)2, Fv, single domain antibody) thereof that competes with theintact antibody for specific binding and includes chimeric, humanized,fully human, and bispecific antibodies. In some cases, antigen-bindingfragments are produced, for example, by recombinant DNA techniques. Inother cases, antigen-binding fragments are produced by enzymatic orchemical cleavage of intact antibodies. Antigen-binding fragmentsinclude Fab, Fab′, F(ab)2, F(ab′)2, Fv, and single-chain antibodies.

Monoclonal antibodies and antibody constructs include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is/are identicalwith or homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity. Chimeric antibodies include “primitized”antibodies comprising variable domain antigen-binding sequences derivedfrom a non-human primate (e.g., Old World Monkey, Ape, etc.) and humanconstant region sequences.

Monoclonal antibodies and antibody constructs include antibodiesreferred to as “human” or “fully human.” The terms “human antibody” and“fully human antibody” each refer to an antibody that has an amino acidsequence of a human immunoglobulin, including antibodies isolated fromhuman immunoglobulin libraries or from animals transgenic for one ormore human immunoglobulins and that do not express endogenousimmunoglobulins; for example, Xenomouse® antibodies and antibodies asdescribed in U.S. Pat. No. 5,939,598.

“Genetically altered antibodies” means antibodies wherein the amino acidsequence has been varied from that of a native antibody. Because of therelevance of recombinant DNA techniques in the generation of antibodies,one need not be confined to the sequences of amino acids found innatural antibodies; antibodies can be redesigned to obtain desiredcharacteristics. The possible variations are many and range from changesto just one or a few amino acids to complete redesign of, for example,the variable and/or constant region. Changes in the constant region, ingeneral, are made in order to improve or alter characteristics, such ascomplement fixation, interaction with membranes and other effectorfunctions, as well as manufacturability and viscosity. Changes in thevariable region can be made to improve antigen binding characteristics.

A “Fab fragment” is comprised of one light chain and the CH1 andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” contains one light chain and one heavy chain thatcontains more of the constant region, between the CH1 and CH2 domains,such that an inter-chain disulfide bond can be formed between two heavychains to form a F(ab′)2 molecule.

A “F(ab′)2 fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the CH1 and CH2domains, such that an inter-chain disulfide bond is formed between twoheavy chains.

“Fv fragment” and “single chain antibody” refer to polypeptidescontaining antibody variable regions from both heavy and light chainsbut lacking constant regions. Like an intact antibody, an Fv fragment orsingle chain antibody are able to bind selectively to a specificantigen. With a molecular weight of only about 25 kD, Fv fragments aremuch smaller than common antibodies (150-160 kD), and even smaller thanFab fragments (about 50 kD, one light chain and half a heavy chain).

A “single domain antibody” is an antibody fragment consisting of asingle domain Fv unit, e.g., VH or VL. Like an intact antibody, a singledomain antibody is able to bind selectively to a specific antigen. Witha molecular weight of only 12-15 kD, single-domain antibodies are muchsmaller than common antibodies (150-160 kD) which are composed of twoheavy polypeptide chains and two light chains, and even smaller than Fabfragments (about 50 kD, one light chain and half a heavy chain) andsingle-chain variable fragments (about 25 kD, two variable domains, onefrom a light and one from a heavy chain). Nanobodies derived from lightchains have also been shown to bind specifically to target epitopes.

“Amino acid” refers to either natural and/or unnatural or syntheticamino acids, including glycine and both the D and L optical isomers,amino acid analogs and peptidomimetics. In some aspects, the term aminoacid refers to monomeric amino acids.

“Clips” are fragments of a polypeptide, such as of a therapeuticpolypeptide. “Clipping” can occur by chemical or enzymatic means. Clipscan range in size, for example, from 0.3 kD to about 50 kD, such as, inkD, about 0.3, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, and any intervening value.Alternatively, clips can be about 3 to about 455 amino acids, such asfrom about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,370, 380, 390, 400, 410, 420, 430, 440, 450, to about 455 amino acids.

A “sample” refers to a composition comprising at least a therapeuticpolypeptide. Therapeutic polypeptides are discussed below. Samples cancomprise additional components, including fragments of the therapeuticpolypeptide. For the purposes of the disclosed methods and compositions,samples are in liquid form.

“Fractionating,” “fractionation,” and the like means separating some ofat least one component of a sample from another different component of asample.

The “fractionation range” of a resin for size-exclusion chromatography,is that range (in size) of molecules for which the resin is useful. Forexample, in resolving polypeptides, a resin having a fractionation rangeof 1,000 Da to 5,000 Da means that those polypeptides having a MW of1,000 Da to 5,000 Da are retained by the pores of the resin. The uppervalue of the range is the critical molecular mass that is completelyexcluded from the resin. All solutes in the sample that are equal to, orlarger, than the critical size elute in the excluded volume of a columnpacked with the resin. The lower value of the range is the criticalmolecular mass that is completely included within the pores of theresin. All solutes that are equal to, or smaller, than this criticalsize behave identically: they elute in the included volume of thecolumn. Solutes between these two ranges of molecular mass elute betweenthe excluded and included volumes.

“Low molecular weight” (LMW) species, when referring to polypeptides,refer to those polypeptides that are smaller the nominal MW of theexamined (target) polypeptide. A LMW fraction is a fraction of aseparation that comprises LMW species. Thus, for example, if a targetpolypeptide is 200 kD, then LMW species have a MW less than 200 kD, suchas for example, (in kD), 199, 195, 190, 185, 180, 175, 170, 165, 160,155, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10,5, and 1 and less and any intervening value. In another example, a LMWspecies for a target polypeptide having a MW of 50 kD, then LMW speciesinclude those that are less than 50 kD, such as (in kD) 49, 45, 40, 35,30, 25, 20, 15, 10, 5, and 1, and less and any intervening value

“High molecular weight” (HMW) species, when referring to polypeptides,refer to those polypeptides that are larger than the nominal MW of theexamined (target) polypeptide. A HMW fraction is a fraction of aseparation that comprises HMW species. Thus, for example, if a targetpolypeptide is 200 kD, then HMW species have a MW greater than 200 kD,such as for example (in kD), 201, 205, 210, 220, 230, 240, 250, 260,270, 280, 290, 300, 325, 350, 375, 400, 450, 500, 550, 600, 700, 800,900, and 1000, and greater and any intervening value. In anotherexample, a HMW species of a target polypeptide having a MW of 50 kDincludes those polypeptides that have a MW greater than 50 kD,including, for example (in kD) 51, 55, 60, 75, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 325, 350, 375, 400, 450, 500, 550, 600, 700, 800, 900, and1000, and greater and any intervening value.

The MW of a target polypeptide can be calculated from the amino acidsequence, provided that the polypeptide is not modified, e.g, such as bypost-translational modifications, phosphorylation, the charge of thepolypeptide at a given pH, or other modification. In the case where anaccurate MW of the target polypeptide cannot be calculated from theamino acid sequence or the sequence is unknown, one of skill in the artcan choose from a variety of methods to determine the MW, including, forexample, size-exclusion chromatography (SEC), Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis (SDS-PAGE), two-dimensional gelelectrophoresis, rate-zonal centrifugation through a density gradient(e.g., sucrose gradient), and time-of-flight mass spectrometry. Whencalculation of MW using sequence is unavailable or inaccurate, SEC ispreferred.

Methods

As previously discussed and referring to FIG. 1, a parental polypeptideand fragments thereof (“clips”) are separated by gel filtration or sizeexclusion-type columns 1, 2. Before this initial fractionation, theparental polypeptide and clips can be treated to, for example,facilitate detection, separation, etc. For example, the polypeptide andclips can be denatured (e.g., disrupting a polypeptide's secondary,tertiary, and quaternary structure from its native state), aided by, forexample, reduction, which disrupts disulfide bonds, and alkylation ofthiols (thus preventing the reforming of disulfide bonds), thusmaintaining the proteins in their unfolded state. High molecular weightspecies elute earlier and are collected first 3. The high molecularweight fraction is then digested with one or more proteolytic enzymes 4.Proteolytic enzymes, such as trypsin, cleave polypeptides. The lowmolecular weight species fraction 2, 5, which usually contains anyclips, elutes later and can be collected separately 6, 7. The lowmolecular weight fraction is evaluated to determine if the clips are anappropriate size for analysis 8. If the clips in this fraction are anappropriate size, then the fraction can be analyzed, such as by usingmass spectrometry, separately from the high molecular weight fraction 2,or mixed with the digested high molecular weight fraction 9 and thenanalyzed. However, if the clips are too large for the intendedanalytical method(s), then this fraction is also digested with one ormore proteolytic enzymes 10. Again, the digested high molecular weightfraction and the digested low molecular weight fraction can be analyzedseparately, or combined 11. Analysis of combined samples is simple andfavors clip quantitation because the clips are analyzed together withthe remaining counterpart of the parental polypeptide.

Method Steps and Components of the Disclosed Compositions

Polypeptide Preparation

The parental polypeptide and associated clips, if any, of a sample canbe optionally prepared for analysis using techniques well known to thoseof skill in the art. Specifically, denaturation of the polypeptides canbe advantageous before any fractionation or analytical steps areperformed.

Protein denaturation can be accomplished by any means that disruptsquaternary, tertiary, or secondary polypeptide structure. For example,the use of chaotropes, such as urea, and denaturing detergents (e.g.,sodium dodecyl sulfate (SDS)), heat, reducing agents, and agents thatinactivate reactive thiol groups to block disulfide reformation. The pHof polypeptide-containing samples can also be manipulated to encouragedenaturation. These components are often used together to effectivelyunfold polypeptides.

Additional examples of chaotropes include, in addition to urea,n-butanol, ethanol, guanidinium chloride, lithium perchlorate, lithiumacetate, magnesium chloride, phenol, 2-propanol, and thiourea. Urea ispreferred in most instances.

Detergents are classified in the form of the hydrophilic group: anionic,cationic, non-ionic, and zwitterionic. Anionic and cationic detergentsare more likely to be denaturing, examples of which include: SDS, sodiumcholate, sodium deoxycholate, sodium glycocholate, sodium taurocholate,sodium taurodeoxycholate, N-lauroylsarcosine, lithium dodecyl sulfate(anionic) and hexadecyltrimethyl ammonium bromide (CTAB) andtrimethyl(tetradecyl) ammonium bromide (TTAB) (cationic). In some cases,a zwitterionic detergent can be useful, examples includeamidosulfobetaine-14 (ASB-14), amidosulfobetaine-16 (ASB-16), C7Bz0,CHAPS, CHAPSO, EMPIGEN® BB, 3-(N,N-dimethyloctylammonio)propanesulfonateinner salt (SB3-8), d (decyldimethylammonio) propanesulfonate inner salt(SB3-10), etc. Anionic detergents are preferred, with SDS beingparticularly preferred.

A “reducing agent”, “reductant” or “reducer” is an element or compoundthat loses (or donates) an electron to another chemical species in aredox chemical reaction. A reducing agent allows disulfide groups tobecome reactive by generating thiol (—SH) groups. Examples of reducingagent include glutathione, β-mercaptoethanol (β-ME), dithiothreitol(DTT), and tris(2-carboxyethyl)phosphine (TCEP).

“Inactivating reactive thiol groups” means blocking free thiol groups ina polypeptide to prevent unwanted thiol-disulfide exchange reactions.Alkylating agents are substances that cause the replacement of hydrogenby an alkyl group. Examples of alkylating agents includeN-ethylmaleimide (NEM), iodoacetamide, and iodoacetic acid; otheralkylating reagents also include acrylamide, 2-vinylpyridine,4-vinylpyridine. Iodoacetamide is usually preferred.

Fractionation

Fractionation is a method suitable for separating polypeptides intodifferent fractions based on one or more characteristics of theseparated polypeptides. Useful for the disclosed methods is theapplication of size exclusion chromatography (SEC), wherein polypeptidesare separate based principally on their molecular size (a function ofmolecular weight (kD) and the number of amino acid residues). Suchmethods use matrices (resins) that can retard proteins of a range ofmolecular sizes while letting other sizes pass through more quickly.

Most usefully, the matrix is packed into a column, which uses gravityflow and/or centrifugation-assisted flow (using “spin” devices) ofsolutions through the matrix in the column. A filter can be used toretain the matrix such that the eluate is collected in the bottom ofspin devices. In some instances, “spin cup” devices are used, while inother instances, many samples can be simultaneously separated using spinplates, such as 96-well microtiter plates.

Gel filtration, or SEC when such procedures are run using aqueoussolvents, is a classic method for fractionating molecules by size.Separation can be regarded as a result of different retention time ofsolutes in the liquid phase when trapped by the porous SEC matrix.Retention is related to the fraction of the pores accessible to thesolute. A thorough review is presented by Hagel (2011. Proteinpurification: Principles, high resolution methods, and applications.3^(rd) ed. Ed. J-C. Janson. John Wiley & Sons, Inc. pp. 51-91).

Considerations for SEC methods include pH, solvent, ionic strength, theuse of any additives, and possible pre-treatments. Furthermore, the SECmatrix is selected not only to be compatible with these variables, butto also have the appropriate separation range; adsorption onto thematrix, such as by the target solutes, needs to also be considered (andpreferably eliminated to obtain a separation based only on size).Extensive optimizations may be necessary depending on the nature of thesample (target polypeptide analyte, impurities) and resolution and timerequirements. In addition to the SEC matrix, column dimensions (if usinga column), matrix packing efficiency, and running conditions can alleffect the results.

To normalize polypeptides to avoid ambiguities in SEC, polypeptides arepreferably denatured and sulfhydryl groups modified so as to preventre-folding of the polypeptides (as described above). When ionicdetergents are used as part of the denaturing protocol, such detergentsbind the polypeptides, resulting in complexes that are substantiallylarger than the polypeptide itself. Furthermore, charged groups from theionic detergent(s) are also introduced. Ionic conditions and pH playimportant roles in polypeptide size because of the charged nature ofpolypeptides. These attributes factor into the choice of SEC matrix(specifically, the appropriate pore size contained by the particles ofthe matrix).

The selection of SEC matrix is first generally made based on theintended solvent and pH. Characteristics of selected SEC matrices isshown in Table A.

TABLE A SEC supports for polypeptide separation* Fractionation pHSupport type Matrix Type range (kD) stability Dextran Sephadex ®¹ G-501.5-30   2-10 G-75 3-80  G-100 4-100 G-150 5-300 Agarose Sepharose ®¹ 6B10-4000 4-9  4B  60-20,000 2B  70-40,000 Sepharose ® 6B 10-4000 3-14 CL¹4B  60-20,000 2B  70-40,000 Ultrogel ®² A6 25-2400 3-10 A4 55-9000 A2 120-23,000 Bio-Gel ®³ A-0.5m 1-500 4-13 A-1.5m  1-1500 A-5m 10-50001-15m  40-15,000 A-50m  100-50,000 Polyacrylamide Bio-Gel ®³ P-101.5-20   2-10 P-30 2.5-40   P-60 3-60  P-100 5-100 P-150 15-150  P-20030-200  P-300 60-400  Dextran/ Sephacryl ®¹ S-200 HR 5-250 2-11bisacrylamide S-300 HR 10-1500 S-400 HR 20-8000 Agarose/ Ultrogel ®² AcA202 1-15  3-10 acrylamide AcA 54 5-70  AcA 44 10-130  AcA 34 20-350  AcA22 100-1200  *After Hagel 2011. Protein purification: Principles, highresolution methods, and applications. 3^(rd) ed. Ed. J-C. Janson. JohnWiley & Sons, Inc. pp. 51-91 ¹GE Healthcare ²Pall Corporation ³Bio-RadLaboratories

To apply polypeptides to a SEC matrix and collect them, elution bufferscan be used (“mobile phase”). In general, characteristics to beconsidered with such buffers include pH, salt concentration, and, ofcourse, appropriate buffers (e.g., those that buffer well at the desiredpH). In some cases, a detergent and/or a solvent can also be added tothe elution buffer. Furthermore, amino acids, such as methionine, can beadded to prevent artefactual oxidation or other artifacts of samplepreparation. Preferably, the elution buffer contains buffer and/or saltto overcome ionic interactions, but not so much to cause hydrophobicinteractions and does not alter the polypeptides.

Buffer composition is carefully chosen, being cognizant that buffers canaffect the shape or biological activity of the molecules. Extremes of pHand ionic strength and denaturing agents can cause conformationalchanges and dissociation or association of protein complexes. Regardingbuffer concentration, a concentration is selected that maintainsbuffering capacity and a constant pH during the procedure. Toprevent/disrupt non-specific ionic interaction with the matrix, a saltcan be used, such as NaCl, up to, for example 300 mM. Preferably,solutions used in SEC applications are filtered to remove sub-micronparticles (e.g., solutions are filtered through 0.45 μm or 0.22 μmmembranes before use). When running SEC columns under atmosphericconditions, solutions are preferably degassed. A useful initial buffercondition that can be used to start optimizing conditions for aparticular polypeptide analyte (or set of polypeptide analytes) is 50 mMsodium phosphate, 150 mM sodium chloride, pH 7.0. Another example of auseful buffer is 100 mM Tris, 50 mM methionine, pH 7.5.

Samples are eluted isocratically (i.e., without changing the compositionof the mobile phase) from a SEC column, using a single buffer system. Inthe disclosed methods, at least two elution steps are performed; a firstelution and a second elution, wherein the first elution collects large(“parental”) polypeptides (the high molecular weight fraction), and thesecond elution harvests polypeptide fragments (clips) of the larger,parental polypeptides (the low molecular weight fraction).

Peptide Preparation

In preparation for analysis, such as by the mass spectrometry-basedmulti-attribute method (e.g., Rogers, et al. 2015. Development of aquantitative mass spectrometry multi-attribute method forcharacterization, quality control testing and disposition of biologics.mAbs. 7:5, 881-890), the eluted polypeptides can be fragmented.Enzymatic and non-enzymatic cleavage agents are shown in Table B.

TABLE B Polypeptide cleavage reagents* Reagent Cleavage site Optimal pHEnzymatic chymotrypsin carboxy side of Tyr, Trp, Phe, 7-9 Leuclostripain carobxy side of Arg (and Lys, 7.7 less frequently) elastasecarboxy side of uncharged 7-9 aliphatic residues endoproteinase carboxyside of Asp and Glu 7.8 (at pH 4, only Arg-C (except if Glu is within2-3 cleaves at Glu) residues of N-terminus) Endoproteinase amino side ofAsp 6.5-8.5 (cleaves amino Asp-N side of cysteine if (flavastacin)cysteine is not reduced) Endoproteinase carboxy side of Glu and Asp 4-8(cleaves only Glu-C at Glu at pH 4) endoproteinase carboxy side of Lys8.5-8.8 Lys-C kallikrein carboxy side of Arg, 7-8 preferable if theresidue just amino acid is Phe or Leu) papain amino side of any amino6-7 acid, but preferentially if immediately amino amino acid is Arg,Lys, Gln, His, Gly, or Tyr pepsin A amino side of any 1.8-2.2hydrophobic amino acid, but preferentially if the immediately aminoamino acid is Phe, Met, Leu, or Tryp plasmin carboxy side of Arg, Lys8.9 thermolysin amino side of hydrophobic 7-9 amino acid, lessfrequently however if Gly, Ser, Thre and not if carboxy amino acid isPro thrombin carboxy side of Arg 8.2-9   trypsin carboxy side of Arg,Lys 8.2-9   V8 protease carboxy side of Glu, Asp 8.5 V8 protease carboxyside of Glu 6-7 Chemical cyanogen carboxy side of Met buffer: 70%formate bromide BNPS-skatole Carboxy side of Trp buffer: 50% glacialacetic acid formic acid Between Asp and Pro buffer: 88% in waterchloramine T Carboxy side fo Trp buffer: water *Excerpted and modifiedfrom Judd, 1994. “Comparison of protein primary structures. Peptidemapping.” Methods Mol Biol. 32: 185-205 and Smith, 1994. “Enzymaticmethods for cleaving proteins.” Methods Mol Biol. 32: 289-296.

After the samples have been processed for peptide fragmentation, theycan be optionally (and preferably) cleaned of the digestion buffers (by,for example, buffer exchange) and of salts and enzymes (e.g., adesalting procedure) before detecting peptide fragments.

Detection Methods and Detectors

Detectors can be a mass spectrometer, a protein imaging device, anenzyme-linked immunosorbent assays (ELISA), or even a protein gel (e.g.,SDS-polyacrylamide gel electrophoresis (SDS-PAGE)). Preferably, massspectrometers are used.

Mass spectrometry refers generally to an analytical technique fordetermining the elemental composition of a sample or molecule.Importantly, mass spectrometry can also be used for determining thechemical structures of molecules, such as those of polypeptides.Commonly, polypeptides ranging from about 0.3 kD to about 8 kD caneasily be analyzed by mass spectrometry; however, as the technologycontinues to advance, polypeptides smaller than 0.3 kD and much largerthan 8 kD can be analyzed.

The principle enabling mass spectrometry (MS) consists of ionizingchemical compounds to generate charged molecules or molecule fragments,and then measuring their mass-to-charge ratios. In an illustrative MSprocedure, a sample is loaded onto the MS instrument and undergoesvaporization, the components of the sample are ionized by one of avariety of methods (e.g., by impacting them with an electron beam),which results in the formation of positively charged particles, thepositive ions are then accelerated by a magnetic field, computations areperformed on the mass-to-charge ratio (m/z) of the particles based onthe details of motion of the ions as they transit throughelectromagnetic fields, and, detection of the ions, which have beensorted according to their m/z ratios.

An illustrative MS instrument has three modules: an ion source, whichconverts gas phase sample molecules into ions (or, in the case ofelectrospray ionization, move ions that exist in solution into the gasphase); a mass analyzer, which sorts the ions by their masses byapplying electromagnetic fields; and a detector, which measures thevalue of an indicator quantity and thus provides data for calculatingthe abundances of each ion present.

The MS technique has both qualitative and quantitative uses, includingidentifying unknown compounds, determining the isotopic composition ofelements in a molecule, and determining the structure of a compound byobserving its fragmentation. Included are gas chromatography-massspectrometry (GC/MS or GC-MS), liquid chromatography mass spectrometry(LC/MS or LC-MS), and ion mobility spectrometry/mass spectrometry(IMS/MS or IMMS).

As shown in the flow of FIG. 1, before analyzing the sample, such as MS,the low molecular weight fraction (fragmented or non-fragmented) can becombined with the high molecular weight (9 or 11) fraction that has beenfragment (using the same fragmentation approach as the low molecularweight fraction, if fragmented). The mixed sample is then used foranalysis. Alternatively, the high and low molecular weight fractions areanalyzed individually.

Therapeutic Polypeptides

Proteins, including those that bind to one or more of the following, canbe useful in the disclosed compositions and methods. These include CDproteins, including CD3, CD4, CD8, CD19, CD20, CD22, CD30, and CD34;including those that interfere with receptor binding. HER receptorfamily proteins, including HER2, HER3, HER4, and the EGF receptor. Celladhesion molecules, for example, LFA-I, Mol, p150, 95, VLA-4, ICAM-I,VCAM, and alpha v/beta 3 integrin. Growth factors, such as vascularendothelial growth factor (“VEGF”), growth hormone, thyroid stimulatinghormone, follicle stimulating hormone, luteinizing hormone, growthhormone releasing factor, parathyroid hormone, Mullerian-inhibitingsubstance, human macrophage inflammatory protein (MIP-1-alpha),erythropoietin (EPO), nerve growth factor, such as NGF-beta,platelet-derived growth factor (PDGF), fibroblast growth factors,including, for instance, aFGF and bFGF, epidermal growth factor (EGF),transforming growth factors (TGF), including, among others, TGF-α andTGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5, insulin-likegrowth factors-I and -II (IGF-I and IGF-II), des(I-3)-IGF-I (brainIGF-I), and osteoinductive factors. Insulins and insulin-relatedproteins, including insulin, insulin A-chain, insulin B-chain,proinsulin, and insulin-like growth factor binding proteins. Coagulationand coagulation-related proteins, such as, among others, factor VIII,tissue factor, von Willebrands factor, protein C, alpha-1-antitrypsin,plasminogen activators, such as urokinase and tissue plasminogenactivator (“t-PA”), bombazine, thrombin, and thrombopoietin; (vii) otherblood and serum proteins, including but not limited to albumin, IgE, andblood group antigens. Colony stimulating factors and receptors thereof,including the following, among others, M-CSF, GM-CSF, and G-CSF, andreceptors thereof, such as CSF-1 receptor (c-fms). Receptors andreceptor-associated proteins, including, for example, flk2/flt3receptor, obesity (OB) receptor, LDL receptor, growth hormone receptors,thrombopoietin receptors (“TPO-R,” “c-mpl”), glucagon receptors,interleukin receptors, interferon receptors, T-cell receptors, stem cellfactor receptors, such as c-Kit, and other receptors. Receptor ligands,including, for example, OX40L, the ligand for the OX40 receptor.Neurotrophic factors, including bone-derived neurotrophic factor (BDNF)and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6). RelaxinA-chain, relaxin B-chain, and prorelaxin; interferons and interferonreceptors, including for example, interferon-α, -β, and -γ, and theirreceptors. Interleukins and interleukin receptors, including IL-I toIL-33 and IL-I to IL-33 receptors, such as the IL-8 receptor, amongothers. Viral antigens, including an AIDS envelope viral antigen.Lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lungsurfactant, tumor necrosis factor-alpha and -beta, enkephalinase, RANTES(regulated on activation normally T-cell expressed and secreted), mousegonadotropin-associated peptide, DNAse, inhibin, and activin. Integrin,protein A or D, rheumatoid factors, immunotoxins, bone morphogeneticprotein (BMP), superoxide dismutase, surface membrane proteins, decayaccelerating factor (DAF), AIDS envelope, transport proteins, homingreceptors, addressins, regulatory proteins, immunoadhesins, antibodies.Myostatins, TALL proteins, including TALL-I, amyloid proteins, includingbut not limited to amyloid-beta proteins, thymic stromal lymphopoietins(“TSLP”), RANK ligand (“OPGL”), c-kit, TNF receptors, including TNFReceptor Type 1, TRAIL-R2, angiopoietins, and biologically activefragments or analogs or variants of any of the foregoing.

Exemplary polypeptides and antibodies include Activase® (Alteplase);alirocumab, Aranesp® (Darbepoetin-alfa), Epogen® (Epoetin alfa, orerythropoietin); Avonex® (Interferon β-Ia); Bexxar® (Tositumomab);Betaseron® (Interferon-β); bococizumab (anti-PCSK9 monoclonal antibodydesignated as L1L3, see U58080243); Campath® (Alemtuzumab); Dynepo®(Epoetin delta); Velcade® (bortezomib); MLN0002 (anti-α4β7 mAb); MLN1202(anti-CCR2 chemokine receptor mAb); Enbrel® (etanercept); Eprex®(Epoetin alfa); Erbitux® (Cetuximab); evolocumab; Genotropin®(Somatropin); Herceptin® (Trastuzumab); Humatrope® (somatropin [rDNAorigin] for injection); Humira® (Adalimumab); Infergen® (InterferonAlfacon-1); Natrecor® (nesiritide); Kineret® (Anakinra), Leukine®(Sargamostim); LymphoCide® (Epratuzumab); Benlysta™ (Belimumab);Metalyse® (Tenecteplase); Mircera® (methoxy polyethylene glycol-epoetinbeta); Mylotarg® (Gemtuzumab ozogamicin); Raptiva® (efalizumab); Cimzia®(certolizumab pegol); Soliris™ (Eculizumab); Pexelizumab (Anti-C5Complement); MEDI-524)(Numax®; Lucentis® (Ranibizumab);Edrecolomab)(Panorex®; Trabio® (lerdelimumab); TheraCim hR3(Nimotuzumab); Omnitarg (Pertuzumab, 2C4); Osidem® (IDM-I); OvaRex®(B43.13); Nuvion® (visilizumab); Cantuzumab mertansine (huC242-DMI);NeoRecormon® (Epoetin beta); Neumega® (Oprelvekin); Neulasta® (Pegylatedfilgastrim, pegylated G-CSF, pegylated hu-Met-G-CSF); Neupogen®(Filgrastim); Orthoclone OKT3® (Muromonab-CD3), Procrit® (Epoetin alfa);Remicade® (Infliximab), Reopro® (Abciximab), Actemra® (anti-IL6 ReceptormAb), Avastin® (Bevacizumab), HuMax-CD4 (zanolimumab), Rituxan®(Rituximab); Tarceva® (Erlotinib); Roferon-A®-(Interferon alfa-2a);Simulect® (Basiliximab); Stelara™ (Ustekinumab); Prexige® (lumiracoxib);Synagis® (Palivizumab); 146B7-CHO (anti-IL15 antibody, see U.S. Pat. No.7,153,507), Tysabri® (Natalizumab); Valortim® (MDX-1303, anti-B.anthracia Protective Antigen mAb); ABthrax™; Vectibix® (Panitumumab);Xolair® (Omalizumab), ETI211 (anti-MRSA mAb), IL-I Trap (the Fc portionof human IgGI and the extracellular domains of both IL-I receptorcomponents (the Type I receptor and receptor accessory protein)), VEGFTrap (Ig domains of VEGFR1 fused to IgGI Fc), Zenapax® (Daclizumab);Zenapax® (Daclizumab), Zevalin® (Ibritumomab tiuxetan), Zetia(ezetimibe), Atacicept (TACI-Ig), anti-α4β7 mAb (vedolizumab); galiximab(anti-CD80 monoclonal antibody), anti-CD23 mAb (lumiliximab); BR2-Fc(huBR3/huFc fusion protein, soluble BAFF antagonist); Simponi™(Golimumab); Mapatumumab (human anti-TRAIL Receptor-1 mAb); Ocrelizumab(anti-CD20 human mAb); HuMax-EGFR (zalutumumab); M200 (Volociximab,anti-α5β1 integrin mAb); MDX-010 (Ipilimumab, anti-CTLA-4 mAb andVEGFR-I (IMC-18F1); anti-BR3 mAb; anti-C. difficile Toxin A and Toxin BC mAbs MDX-066 (CDA-I) and MDX-1388); anti-CD22 dsFv-PE38 conjugates(CAT-3888 and CAT-8015); anti-CD25 mAb (HuMax-TAC); anti-TSLPantibodies; anti-TSLP receptor antibody (U.S. Pat. No. 8,101,182);anti-TSLP antibody designated as A5 (U.S. Pat. No. 7,982,016); (anti-CD3mAb (NI-0401); Adecatumumab (MT201, anti-EpCAM-CD326 mAb); MDX-060,SGN-30, SGN-35 (anti-CD30 mAbs); MDX-1333 (anti-IFNAR); HuMax CD38(anti-CD38 mAb); anti-CD40L mAb; anti-Cripto mAb; anti-CTGF IdiopathicPulmonary Fibrosis Phase I Fibrogen (FG-3019); anti-CTLA4 mAb;anti-eotaxinl mAb (CAT-213); anti-FGF8 mAb; anti-ganglioside GD2 mAb;anti-sclerostin antibodies (see, U.S. Pat. No. 8,715,663 or U.S. Pat.No. 7,592,429) anti-sclerostin antibody designated as Ab-5 (U.S. Pat.No. 8,715,663 or U.S. Pat. No. 7,592,429); anti-ganglioside GM2 mAb;anti-GDF-8 human mAb (MYO-029); anti-GM-CSF Receptor mAb (CAM-3001);anti-HepC mAb (HuMax HepC); MEDI-545, MDX-1103 (anti-IFNα mAb);anti-IGFIR mAb; anti-IGF-IR mAb (HuMax-Inflam); anti-IL12/IL23p40 mAb(Briakinumab); anti-IL-23p19 mAb (LY2525623); anti-IL13 mAb (CAT-354);anti-IL-17 mAb (AIN457); anti-IL2Ra mAb (HuMax-TAC); anti-IL5 ReceptormAb; anti-integrin receptors mAb (MDX-018, CNTO 95); anti-IPIOUlcerative Colitis mAb (MDX-1100); anti-LLY antibody; BMS-66513;anti-Mannose Receptor/hCGβ mAb (MDX-1307); anti-mesothelin dsFv-PE38conjugate (CAT-5001); anti-PDImAb (MDX-1 106 (ONO-4538)); anti-PDGFRaantibody (IMC-3G3); anti-TGFβ mAb (GC-1008); anti-TRAIL Receptor-2 humanmAb (HGS-ETR2); anti-TWEAK mAb; anti-VEGFR/Flt-1 mAb; anti-ZP3 mAb(HuMax-ZP3); NVS Antibody #1; NVS Antibody #2; and an amyloid-betamonoclonal antibody comprising sequences, SEQ ID NO:8 and SEQ ID NO:6 ofU.S. Pat. No. 7,906,625.

Examples of antibodies suitable for the methods and pharmaceuticalformulations include the antibodies shown in Table 1. Other examples ofsuitable antibodies include infliximab, bevacizumab, cetuximab,ranibizumab, palivizumab, abagovomab, abciximab, actoxumab, adalimumab,afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518,alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab mafenatox,anrukinzumab, apolizumab, arcitumomab, aselizumab, altinumab, atlizumab,atorolimiumab, tocilizumab, bapineuzumab, basiliximab, bavituximab,bectumomab, belimumab, benralizumab, bertilimumab, besilesomab,bevacizumab, bezlotoxumab, biciromab, bivatuzumab, bivatuzumabmertansine, blinatumomab, blosozumab, brentuximab vedotin, briakinumab,brodalumab, canakinumab, cantuzumab mertansine, cantuzumab mertansine,caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49,cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox,cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan,conatumumab, crenezumab, cr6261, dacetuzumab, daclizumab, dalotuzumab,daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox,drozitumab, duligotumab, dupilumab, ecromeximab, eculizumab, edobacomab,edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab,enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab,epitumomab cituxetan, epratuzumab, erenumab, erlizumab, ertumaxomab,etaracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab,faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab,ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab,foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab,gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab,girentuximab, glembatumumab vedotin, golimumab, gomiliximab, gs6624,ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab,imgatuzumab, inclacumab, indatuximab ravtansine, infliximab,intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab,itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab,lemalesomab, lerdelimumab, lexatumumab, libivirumab, ligelizumab,lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab,lumiliximab, mapatumumab, maslimomab, mavrilimumab, matuzumab,mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab,mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox,muromonab-cd3, nacolomab tafenatox, namilumab, naptumomab estafenatox,narnatumab, natalizumab, nebacumab, necitumumab, nerelimomab,nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab,ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab,onartuzumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab,oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab,panitumumab, panobacumab, parsatuzumab, pascolizumab, pateclizumab,patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab,pidilizumab, pintumomab, placulumab, ponezumab, priliximab, pritumumab,PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ramucirumab,ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab,rituximab, robatumumab, roledumab, romosozumab, rontalizumab,rovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetide,secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab,simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab,sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumabtetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox,tefibazumab, telimomab aritox, tenatumomab, tefibazumab, teneliximab,teplizumab, teprotumumab, TGN1412, tremelimumab, ticilimumab,tildrakizumab, tigatuzumab, TNX-650, tocilizumab, toralizumab,tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab,tucotuzumab celmoleukin, tuvirumab, ublituximab, urelumab, urtoxazumab,ustekinumab, vapaliximab, vatelizumab, vedolizumab, veltuzumab,vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumabmafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab,zolimomab aritox.

Antibodies also include adalimumab, bevacizumab, blinatumomab,cetuximab, conatumumab, denosumab, eculizumab, erenumab, evolocumab,infliximab, natalizumab, panitumumab, rilotumumab, rituximab,romosozumab, tezepelumab, and trastuzumab, and antibodies selected fromTable 1.

TABLE 1 Examples of therapeutic antibodies Target HC Type (informalConc. Viscosity (including LC LC SEQ HC SEQ name) (mg/ml) (cP)allotypes) Type pI ID NO ID NO anti-amyloid 142.2 5.0 IgG1 (f) (R; EM)Kappa 9.0 1 2 GMCSF (247) 139.7 5.6 IgG2 Kappa 8.7 3 4 CGRPR 136.6 6.3IgG2 Lambda 8.6 5 6 RANKL 152.7 6.6 IgG2 Kappa 8.6 7 8 Sclerostin 145.06.7 IgG2 Kappa 6.6 9 10 (27H6) IL-1R1 153.9 6.7 IgG2 Kappa 7.4 11 12Myostatin 141.0 6.8 IgG1 (z) (K; EM) Kappa 8.7 13 14 B7RP1 137.5 7.7IgG2 Kappa 7.7 15 16 Amyloid 140.6 8.2 IgG1 (za) (K; DL) Kappa 8.7 17 18GMCSF (3.112) 156.0 8.2 IgG2 Kappa 8.8 19 20 CGRP (32H7) 159.5 8.3 IgG2Kappa 8.7 21 22 CGRP (3B6.2) 161.1 8.4 IgG2 Lambda 8.6 23 24 PCSK9(8A3.1) 150.0 9.1 IgG2 Kappa 6.7 25 26 PCSK9 (492) 150.0 9.2 IgG2 Kappa6.9 27 28 CGRP 155.2 9.6 IgG2 Lambda 8.8 29 30 Hepcidin 147.1 9.9 IgG2Lambda 7.3 31 32 TNFR p55) 157.0 10.0 IgG2 Kappa 8.2 33 34 OX40L 144.510.0 IgG2 Kappa 8.7 35 36 HGF 155.8 10.6 IgG2 Kappa 8.1 37 38 GMCSF162.5 11.0 IgG2 Kappa 8.1 39 40 Glucagon R 146.0 12.1 IgG2 Kappa 8.4 4142 GMCSF (4.381) 144.5 12.1 IgG2 Kappa 8.4 43 44 Sclerostin 155.0 12.1IgG2 Kappa 7.8 45 46 (13F3) CD-22 143.7 12.2 IgG1 (f) (R; EM) Kappa 8.847 48 INFgR 154.2 12.2 IgG1 (za) (K; DL) Kappa 8.8 49 50 Ang2 151.5 12.4IgG2 Kappa 7.4 51 52 TRAILR2 158.3 12.5 IgG1 (f) (R; EM) Kappa 8.7 53 54EGFR 141.7 14.0 IgG2 Kappa 6.8 55 56 IL-4R 145.8 15.2 IgG2 Kappa 8.6 5758 IL-15 149.0 16.3 IgG1 (f) (R; EM) Kappa 8.8 59 60 IGF1R 159.2 17.3IgG1 (za) (K; DL) Kappa 8.6 61 62 IL-17R 150.9 19.1 IgG2 Kappa 8.6 63 64Dkk1 (6.37.5) 159.4 19.6 IgG2 Kappa 8.2 65 66 Sclerostin 134.8 20.9 IgG2Kappa 7.4 67 68 TSLP 134.2 21.4 IgG2 Lambda 7.2 69 70 Dkk1 (11H10) 145.322.5 IgG2 Kappa 8.2 71 72 PCSK9 145.2 22.8 IgG2 Lambda 8.1 73 74 GIPR150.0 23.0 IgG1 (z) (K; EM) Kappa 8.1 75 76 (2G10.006) Activin 133.929.4 IgG2 Lambda 7.0 77 78 Sclerostin (2B8) 150.0 30.0 IgG2 Lambda 6.779 80 Sclerostin 141.4 30.4 IgG2 Kappa 6.8 81 82 c-fms 146.9 32.1 IgG2Kappa 6.6 83 84 α4β7 154.9 32.7 IgG2 Kappa 6.5 85 86 * An exemplaryconcentration suitable for patient administration; {circumflex over( )}HC—antibody heavy chain; LC—antibody light chain.

EXAMPLES Example 1—Two-Step Elution Procedure to Identify PolypeptideClips

Samples of a mutated glycoprotein cytokine Fc fusion (MGC-1) weredenatured, reduced, and alkylated using standard methods before loadingonto Illustra™ NAP™-5 columns (columns prepacked with Sephadex® G-25 DNAGrade in distilled water containing 0.15% Kathon™ CG/ICP Biocide; GEHealthcare Life Sciences; Piscataway, N.J.).

Denaturing buffer was composed of 7.5 M Guanidine HCl, 250 mM Tris, 2 mMEDTA. Reduction buffer was 0.5 M dithiothreitol (DTT) and alkylationbuffer was 0.5 M sodium iodoacetate (IA). Reduction was carried out for30 minutes and alkylation was done in 20 minutes. The reduced andalkylated MGC-1 samples were loaded into NAP 5 columns and were elutedwith pH 7.5 100 mM Tris buffer containing 50 mM methionine. Theflow-throughs in the elution step were collected in Eppendorf tubes andwere separated by a reversed-phase liquid chromatography (LC) columnbefore being detected by a mass spectrometer.

A two-step elution was used to recover the clips. The first elution waswith 700 μL elution buffer and the whole reduced alkylated MGC-1 waseluted and collected in Fraction 1 as shown in FIG. 2A. This was theintended use of this desalting column and normally after this elution,the desalting column would be discarded (Ren D, et al. 2009. AnalBiochem. 392(1): 12-21.). But what was observed was that the clips thatwere detached from MGC-1 whole molecule were trapped inside thedesalting material of the NAP™-5 column, and these clips were recoveredby eluting with another 400 μL of elution buffer and collected asFraction 2 as shown in FIG. 2B.

Data analysis showed that the major component of Fraction 1 was thewhole reduced and alkylated MGC-1, as expected. Analyses of Fraction 2revealed that peaks eluted at 20 min and 21 min were C-terminal clips(12 amino acids; FIG. 2B, 1) and middle-region clips (51 amino acids,FIG. 2B, 2), respectively. These results demonstrated that the two-stepelution process successfully recovered the lower molecular weight clips.

This methodology can be also used for filter assisted desalting.Normally, the eluent below molecular cutoff, e.g., 10 KDa, arediscarded, thus discarding clips below 10 kD with the salt. This eluentcan be collected and subjected to further analysis using of two stepelution using NAP™-5 columns as outlined in this example.

1. A method of detecting at least one fragment of a polypeptide in asample, comprising: (a) fractionating the sample, comprising: a. loadingthe sample onto a size exclusion chromatographic matrix; b. applying avolume of a first elution buffer to the matrix, wherein the polypeptideis eluted into a first fraction; and c. applying a volume of a secondelution buffer to the matrix, wherein the at least one fragment iseluted into a second fraction; (b) treating the first fraction with aproteolytic enzyme; and (c) optionally, combining the treated firstfraction with the second fraction, and (d) detecting the polypeptide inthe first fraction and detecting the at least one fragment in the secondfraction.
 2. (canceled)
 3. The method of claim 1, further comprisingafter step (a), treating the second fraction with a proteolytic enzyme.4. The method of claim 1, wherein the polypeptide is a therapeuticpolypeptide.
 5. The method of claim 4, wherein the therapeuticpolypeptide is selected from the group consisting of an antibody orantigen-binding fragment thereof, a derivative of an antibody orantibody fragment, and a fusion polypeptide.
 6. The method of claim 5,wherein the antibody is selected from the group consisting ofinfliximab, bevacizumab, cetuximab, ranibizumab, palivizumab,abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab,alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab,altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab,arcitumomab, aselizumab, altinumab, atlizumab, atorolimiumab,tocilizumab, bapineuzumab, basiliximab, bavituximab, bectumomab,belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab,bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine,blinatumomab, blosozumab, brentuximab vedotin, briakinumab, brodalumab,canakinumab, cantuzumab mertansine, cantuzumab mertansine, caplacizumab,capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab,certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab,clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab,crenezumab, cr6261, dacetuzumab, daclizumab, dalotuzumab, daratumumab,demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab,duligotumab, dupilumab, ecromeximab, eculizumab, edobacomab,edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab,enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab,epitumomab cituxetan, epratuzumab, erenumab, erlizumab, ertumaxomab,etaracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab,faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab,ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab,foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab,gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab,girentuximab, glembatumumab vedotin, golimumab, gomiliximab, gs6624,ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab,imgatuzumab, inclacumab, indatuximab ravtansine, infliximab,intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab,itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab,lemalesomab, lerdelimumab, lexatumumab, libivirumab, ligelizumab,lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab,lumiliximab, mapatumumab, maslimomab, mavrilimumab, matuzumab,mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab,mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox,muromonab-cd3, nacolomab tafenatox, namilumab, naptumomab estafenatox,narnatumab, natalizumab, nebacumab, necitumumab, nerelimomab,nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab,ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab,onartuzumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab,oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab,panitumumab, panobacumab, parsatuzumab, pascolizumab, pateclizumab,patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab,pidilizumab, pintumomab, placulumab, ponezumab, priliximab, pritumumab,PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ramucirumab,ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab,rituximab, robatumumab, roledumab, romosozumab, rontalizumab,rovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetide,secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab,simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab,sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumabtetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox,tefibazumab, telimomab aritox, tenatumomab, tefibazumab, teneliximab,teplizumab, teprotumumab, tezepelumab, TGN1412, tremelimumab,ticilimumab, tildrakizumab, tigatuzumab, TNX-650, tocilizumab,toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07,tregalizumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, urelumab,urtoxazumab, ustekinumab, vapaliximab, vatelizumab, vedolizumab,veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab,vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab,ziralimumab, zolimomab aritox, and those antibodies shown in the tablebelow: Light chain Heavy chain Target (informal name) SEQ ID NO SEQ IDNO anti-amyloid 1 2 GMCSF (247) 3 4 CGRPR 5 6 RANKL 7 8 Sclerostin(27H6) 9 10 IL-1R1 11 12 Myostatin 13 14 B7RP1 15 16 Amyloid 17 18 GMCSF(3.112) 19 20 CGRP (32H7) 21 22 CGRP (3B6.2) 23 24 PCSK9 (8A3.1) 25 26PCSK9 (492) 27 28 CGRP 29 30 Hepcidin 31 32 TNFR p55) 33 34 OX40L 35 36HGF 37 38 GMCSF 39 40 Glucagon R 41 42 GMCSF (4.381) 43 44 Sclerostin(13F3) 45 46 CD-22 47 48 INFgR 49 50 Ang2 51 52 TRAILR2 53 54 EGFR 55 56IL-4R 57 58 IL-15 59 60 IGF1R 61 62 IL-17R 63 64 Dkk1 (6.37.5) 65 66Sclerostin 67 68 TSLP 69 70 Dkk1 (11H10) 71 72 PCSK9 73 74 GIPR(2G10.006) 75 76 Activin 77 78 Sclerostin (2B8) 79 80 Sclerostin 81 82c-fms 83 84 α4β7 85 86


7. The method of claim 4, wherein the therapeutic polypeptide is apolypeptide selected from the group consisting a glycoprotein, CDpolypeptide, a HER receptor polypeptide, a cell adhesion polypeptide, agrowth factor polypeptide, an insulin polypeptide, an insulin-relatedpolypeptide, a coagulation polypeptide, a coagulation-relatedpolypeptide, albumin, IgE, a blood group antigen, a colony stimulatingfactor, a receptor, a neurotrophic factor, an interferon, aninterleukin, a viral antigen, a lipoprotein, calcitonin, glucagon,atrial natriuretic factor, lung surfactant, tumor necrosis factor-alphaand -beta, enkephalinase, mouse gonadotropin-associated peptide, DNAse,inhibin, activing, an integrin, protein A, protein D, a rheumatoidfactor, an immunotoxin, a bone morphogenetic protein, a superoxidedismutase, a surface membrane polypeptide, a decay accelerating factor,an AIDS envelope, a transport polypeptide, a homing receptor, anaddressin, a regulatory polypeptide, an immunoadhesin, a myostatin, aTALL polypeptide, an amyloid polypeptide, a thymic stromallymphopoietin, a RANK ligand, a c-kit polypeptide, a TNF receptor, andan angiopoietin, and biologically active fragments, analogs or variantsthereof.
 8. The method of claim 1, wherein the fragments are 0.3 kD toabout 50 kD.
 9. The method of claim 1, wherein the proteolytic enzymecomprises trypsin.
 10. The method of claim 3, wherein the proteolyticenzyme comprises trypsin.
 11. The method of claim 1, wherein the matrixcomprises a gel filtration matrix.
 12. The method of claim 11, whereinthe gel filtration matrix comprises cross-linked dextran.
 13. The methodof claim 1, wherein the matrix is prepared in a column before performingstep (a).
 14. The method of claim 13, wherein the column is suitable forgravity flow or centrifugal flow.
 15. The method of claim 1, wherein thefirst elution buffer and the second elution buffer are the same.
 16. Themethod of claim 15, wherein the first elution buffer and the secondelution buffer comprise 100 mM Tris and 50 mM methionine, pH 7.5. 17.The method of claim 1, wherein the first elution buffer volume isgreater than the second elution buffer volume.
 18. The method of claim1, further comprising, before step (a), performing at least one stepselected from the group consisting of polypeptide alkylation,polypeptide reduction, and polypeptide denaturation, or any combinationthereof.
 19. The method of claim 1, further comprising detecting thepolypeptide and fragments.
 20. The method of claim 19, wherein thedetecting comprises mass spectrometry.
 21. A sample, prepared by themethod of claim
 1. 22. A method of detecting at least one fragment of aglycoprotein in a sample, comprising: (a) denaturing, reducing, andalkylating the glycoprotein and fragments in the sample; (b)fractionating the sample, comprising, a. loading the sample onto across-linked dextran gel filtration column, wherein the cross-linkeddextran gel has a fractionation range of about 1000-5000 Da; b. applyinga volume of a first elution buffer comprising 100 mM Tris and 50 mMmethionine, pH 7.5 to the column, wherein the glycoprotein is elutedinto a first fraction; and c. applying a volume of second elution buffercomprising 100 mM Tris and 50 mM methionine, pH 7.5 to the column, wherethe at least one fragment is eluted into the second fraction; (c)treating the first fraction with trypsin; (d) combining the treatedfirst fraction with the second fraction; and (e) detecting theglycoprotein in the first fraction and the at least one fragment in thesecond fraction.
 23. The method of claim 22, further comprising, afterstep (b), treating the second fraction with trypsin.
 24. A method ofdetecting at least one fragment of a polypeptide in a sample,comprising: (a) fractionating the sample, comprising: i. loading thesample onto a centrifugal device comprising a molecular weight cutofffilter; and ii. processing the sample such that the sample passesthrough the cutoff filter such that the polypeptide is retained on thefilter to create a first fraction and the at least one fragment thatpasses through the filter is retained as a second fraction; (b) treatingthe first fraction with a proteolytic enzyme; (c) optionally, combiningthe treated first fraction with the second fraction; and (d) detectingthe polypeptide in the first fraction and the fragments in the secondfraction.
 25. The method of claim 24, further comprising after step (a),treating the second fraction with a proteolytic enzyme.
 26. The methodof 24, wherein the polypeptide is a therapeutic polypeptide.
 27. Themethod of claim 26, wherein the therapeutic polypeptide is selected fromthe group consisting of an antibody or antigen-binding fragment thereof,a derivative of an antibody or antibody fragment, and a fusionpolypeptide.
 28. The method of claim 27, wherein the antibody isselected from the group consisting of infliximab, bevacizumab,cetuximab, ranibizumab, palivizumab, abagovomab, abciximab, actoxumab,adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol,ald518, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomabmafenatox, anrukinzumab, apolizumab, arcitumomab, aselizumab, altinumab,atlizumab, atorolimiumab, tocilizumab, bapineuzumab, basiliximab,bavituximab, bectumomab, belimumab, benralizumab, bertilimumab,besilesomab, bevacizumab, bezlotoxumab, biciromab, bivatuzumab,bivatuzumab mertansine, blinatumomab, blosozumab, brentuximab vedotin,briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumabmertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab,cc49, cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox,cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan,conatumumab, crenezumab, cr6261, dacetuzumab, daclizumab, dalotuzumab,daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox,drozitumab, duligotumab, dupilumab, ecromeximab, eculizumab, edobacomab,edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab,enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab,epitumomab cituxetan, epratuzumab, erenumab, erlizumab, ertumaxomab,etaracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab,faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab,ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab,foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab,gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab,girentuximab, glembatumumab vedotin, golimumab, gomiliximab, gs6624,ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab,imgatuzumab, inclacumab, indatuximab ravtansine, infliximab,intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab,itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab,lemalesomab, lerdelimumab, lexatumumab, libivirumab, ligelizumab,lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab,lumiliximab, mapatumumab, maslimomab, mavrilimumab, matuzumab,mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab,mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox,muromonab-cd3, nacolomab tafenatox, namilumab, naptumomab estafenatox,narnatumab, natalizumab, nebacumab, necitumumab, nerelimomab,nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab,ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab,onartuzumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab,oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab,panitumumab, panobacumab, parsatuzumab, pascolizumab, pateclizumab,patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab,pidilizumab, pintumomab, placulumab, ponezumab, priliximab, pritumumab,PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ramucirumab,ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab,rituximab, robatumumab, roledumab, romosozumab, rontalizumab,rovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetide,secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab,simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab,sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumabtetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox,tefibazumab, telimomab aritox, tenatumomab, tefibazumab, teneliximab,teplizumab, teprotumumab, tezepelumab, TGN1412, tremelimumab,ticilimumab, tildrakizumab, tigatuzumab, TNX-650, tocilizumab,toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07,tregalizumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, urelumab,urtoxazumab, ustekinumab, vapaliximab, vatelizumab, vedolizumab,veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab,vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab,ziralimumab, zolimomab aritox, and those antibodies shown in the tablebelow: Light chain Heavy chain Target (informal name) SEQ ID NO SEQ IDNO anti-amyloid 1 2 GMCSF (247) 3 4 CGRPR 5 6 RANKL 7 8 Sclerostin(27H6) 9 10 IL-1R1 11 12 Myostatin 13 14 B7RP1 15 16 Amyloid 17 18 GMCSF(3.112) 19 20 CGRP (32H7) 21 22 CGRP (3B6.2) 23 24 PCSK9 (8A3.1) 25 26PCSK9 (492) 27 28 CGRP 29 30 Hepcidin 31 32 TNFR p55) 33 34 OX40L 35 36HGF 37 38 GMCSF 39 40 Glucagon R 41 42 GMCSF (4.381) 43 44 Sclerostin(13F3) 45 46 CD-22 47 48 INFgR 49 50 Ang2 51 52 TRAILR2 53 54 EGFR 55 56IL-4R 57 58 IL-15 59 60 IGF1R 61 62 IL-17R 63 64 Dkk1 (6.37.5) 65 66Sclerostin 67 68 TSLP 69 70 Dkk1 (11H10) 71 72 PCSK9 73 74 GIPR(2G10.006) 75 76 Activin 77 78 Sclerostin (2B8) 79 80 Sclerostin 81 82c-fms 83 84 α4β7 85 86


29. The method of claim 26, wherein the therapeutic polypeptide is apolypeptide selected from the group consisting a glycoprotein, CDpolypeptide, a HER receptor polypeptide, a cell adhesion polypeptide, agrowth factor polypeptide, an insulin polypeptide, an insulin-relatedpolypeptide, a coagulation polypeptide, a coagulation-relatedpolypeptide, albumin, IgE, a blood group antigen, a colony stimulatingfactor, a receptor, a neurotrophic factor, an interferon, aninterleukin, a viral antigen, a lipoprotein, calcitonin, glucagon,atrial natriuretic factor, lung surfactant, tumor necrosis factor-alphaand -beta, enkephalinase, mouse gonadotropin-associated peptide, DNAse,inhibin, activing, an integrin, protein A, protein D, a rheumatoidfactor, an immunotoxin, a bone morphogenetic protein, a superoxidedismutase, a surface membrane polypeptide, a decay accelerating factor,an AIDS envelope, a transport polypeptide, a homing receptor, anaddressin, a regulatory polypeptide, an immunoadhesin, a myostatin, aTALL polypeptide, an amyloid polypeptide, a thymic stromallymphopoietin, a RANK ligand, a c-kit polypeptide, a TNF receptor, andan angiopoietin, and biologically active fragments, analogs or variantsthereof.
 30. The method of 24, wherein the fragments are 0.3 kD to about50 kD.
 31. The method of 24, wherein the proteolytic enzyme comprisestrypsin.
 32. The method of claim 25, wherein the proteolytic enzymecomprises trypsin.
 33. The method of 24, wherein the matrix comprises agel filtration matrix.
 34. The method of claim 33, wherein the gelfiltration matrix comprises cross-linked dextran.
 35. The method of 24,wherein the matrix is prepared in a column before performing step (a).36. The method of claim 35, wherein the column is suitable for gravityflow or centrifugal flow.
 37. The method of claim 24, wherein the firstelution buffer and the second elution buffer are the same.
 38. Themethod of claim 37, wherein the first elution buffer and the secondelution buffer comprise 100 mM Tris and 50 mM methionine, pH 7.5. 39.The method of 24, wherein the first elution buffer volume is greaterthan the second elution buffer volume.
 40. The method of 24, furthercomprising, before step (a), performing at least one step selected fromthe group consisting of polypeptide alkylation, polypeptide reduction,and polypeptide denaturation, or any combination thereof.
 41. The methodof 24, further comprising detecting the polypeptide and fragments. 42.The method of claim 41, wherein the detecting comprises massspectrometry.
 43. A sample, prepared by the method of 24.